Reducing a pathological blood level of bile acids with a liposome suspension

Abstract

The present disclosure provides a combination of a liposomal suspension, and of albumin, for use as bile acid sequestrants, wherein the use comprises (a) intraperitoneally administering the liposomal suspension to a subject in need thereof; and (b) intravenously or intraperitoneally administering the albumin to the subject; and (c) removing a dialysate containing liposomes and at least one bile acid from the subject, wherein the at least one bile acid comprises cholic acid, glycocholic acid, glycochenodeoxycholic acid or a combination of at least two thereof.

Description

[0001] METHODS OF REDUCING A PATHOLOGICAL BLOOD LEVEL OF BILE ACIDS

[0002] FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to methods of reducing a pathological blood level of bile acids in subjects in need thereof, using a combination of liposomes and albumin.

[0004] BACKGROUND OF THE DISCLOSURE

[0005] In several liver diseases, the flow of bile acids from the liver into the bile is disrupted, resulting in the accumulation of bile acids in the bloodstream.

[0006] Bile acids are a family of acidic sterols, which consist of a steroid nucleus of four fused hydrocarbon rings with polar hydroxy groups. They are amphiphilic molecules, exhibiting an ionized carboxylate or sulfonate group on the side chain contributing to their water solubility. The main biological functions of bile acids are the digestion and absorption of intestinal cholesterol, triglycerides, fatty acids and fat-soluble vitamins, control of bile acid biosynthesis via negative feedback regulatory mechanism, gallbladder motor function, homeostasis of intestinal microbiota, function in immune response, control of gut-liver axis, epithelial cell proliferation and gene expression through epigenetic mechanism.

[0007] Bile acids are synthesized from cholesterol in the liver by two major biosynthetic pathways regulated by negative feedback mechanism.

[0008] In healthy humans, the neutral biosynthetic pathway contributes to around 90% of the bile acid production. After synthesis of primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) from cholesterol in the liver, both bile acids are conjugated to taurine or glycine in a 1 :3 ratio to produce taurocholic acid (TCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), and taurochenodeoxycholic acid (TCDCA), thereby increasing their water solubility and decreasing their toxicity. The taurine and glycine conjugates are secreted into bile and stored in the gallbladder. From there, they are released into the duodenum for food digestion. 85% of the conjugated bile acids are reabsorbed in the duodenum and are recirculated to the liver via the portal vein. The other 15% enter the colon, where the gut microbiota promotes the biotransformation of the primary bile acids into secondary bile acids (deoxycholic acid (DCA), lithocholic acid (LCA)) and tertiary bile acids (ursodeoxycholic acid (UDCA)). Around 50% of deoxycholic acid as well as small amounts of lithocholic acid and ursodeoxycholic acid are reabsorbed so that at the end only 5% of bile acids are lost by excretion via kidneys and feces. These 5% are 0.2 - 0.6 g of bile acids per day, which corresponds to the daily bile acid synthesis. It illustrates that the hepatic bile acid synthesis is regulated by a negative feedback regulatory mechanism by reabsorption of most bile acids back to the liver inhibiting the synthesis there.

[0009] In liver diseases, the acidic biosynthetic pathway becomes predominant, leading to an increased CDCA production. The change in the bile acid pool composition alters the hydrophobicity and therefore, the toxicity of this composition. Cytotoxicity of bile acids is directly related to their hydrophobicity: LCA > DCA > CDCA > CA > UDCA. Accumulation of hydrophobic acids such as CDCA increases the toxicity and is associated with gastrointestinal diseases. Cholestasis is a pathological condition where normal bile flow out of the liver is reduced or disrupted. It is common in acute and chronic liver diseases such as acute-on-chronic liver failure (ACLF). Cholestatic liver injury is induced by accumulation of hydrophobic bile acids, mainly glycochenodeoxycholic acid (GCDCA). The gut-to-liver axis also plays a critical role in the regulation of metabolic homeostasis and in preventing metabolic diseases. Intestinal bile acids are antimicrobial agents, which regulate the gut microbiome homeostasis by controlling bacterial overgrowth. At the same time gut bacteria metabolize bile acids to regulate bile acid composition and hydrophobicity. Alteration in gut microbiota impairs secondary bile acid production protective of gut barrier function. Modifications in the bile acid pool composition towards more hydrophobic bile acids causes higher toxicity for the microbiome, changing the microbiome composition, while bile acid flow obstruction leads to proliferation and overgrowth of bacteria in the gut. In combination this causes intestinal damage leading to bacterial translocation and systemic inflammation.

[0010] Several studies have described a modulation of serum bile acids profile in cirrhotic patients. Primary bile acids glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), taurochenodeoxycholic acid (TCDCA), taurocholic acid (TCA) and chenodeoxycholic acid (CDCA) were found to be upregulated in cirrhotic patients with acute liver decompensation (AD) compared to cirrhotic patients without decompensation (see Table I, below). Moreover, these bile acids and cholic acid were significantly higher in patients with new onset of AD and ACLF compared to those without. Increased serum levels of tauro- and glyco-conjugated bile acids such as glycocholic acid (GCA) and taurocholic acid (TCA) was correlated with ACLF development, and death. Also, an increase in harmful microbiota and decrease in beneficial microbiota was observed in ACLF patients. A reduction of hydrophobicity (increase in ratio of hydrophilic to hydrophobic bile acids) correlates with more beneficial outcome in ACLF. Generally, the accumulation of bile acids in the bloodstream is typically indicative of liver failure. Recent studies have demonstrated a correlation between elevated serum bile acid levels, bacterial infections, and increased mortality rates.

[0011] There is a need for a method for reducing circulating bile acids in subjects in need thereof, and in specific embodiments circulating hydrophobic bile acids.

[0012] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

[0013] SUMMARY OF THE DISCLOSURE

[0014] The present disclosure shows that a combination of liposomes and albumin significantly improves (i.e. additively or synergically) the capture of bile acids (e.g., hydrophobic bile acids such as cholic acid, glycocholic acid, and glycochenodeoxycholic acid) in a peritoneal fluid model. This method is useful in subjects displaying pathologically elevated blood levels of these bile acids, such as subjects with liver impairments (e.g., cirrhotic subject with or without acute decompensation and the more severe form acute-on-chronic liver failure patients (ACLF) subjects). More specifically, in accordance with the present disclosure, there are provided the following items:

[0015] Item 1. Combination of a liposomal suspension, and of albumin, for use as bile acid sequestrants, wherein the use comprises (a) intraperitoneally administering the liposomal suspension to a subject in need thereof; and (b) intravenously or intraperitoneally administering the albumin to the subject; and (c) removing a dialysate containing liposomes and at least one bile acid from the subject, wherein the at least one bile acid comprises cholic acid, glycocholic acid, glycochenodeoxycholic acid or a combination of at least two thereof. In a specific embodiment, the liposomal suspension comprises transmembrane pH gradient liposomes.

[0016] Item 2. The combination for use of item 1 , wherein in (a) the liposomal suspension is in a dose of between about 2 mM to about 10 mM or of between about 2 mM to about 9.5 mM.

[0017] Item 3. The combination for use of item 1 or 2, wherein in (b) the albumin is in a dose of between about 6 mg / mL to about 10 mg / mL, preferably between about 8 mg / mL to about 10 mg / mL, most preferably about 9 mg / mL.

[0018] Item 4. The combination for use of any one of items 1 to 3, wherein (a) and (b) are simultaneous.

[0019] Item 5. The combination for use of any one of items 1 to 4, wherein the administering in (b) is intraperitoneal.

[0020] Item 6. The combination for use of any one of items 1 to 5, wherein the liposomes contain a hydroxy acid, preferably citric acid, most preferably citric acid anhydrous.

[0021] Item 7. The combination for use of item 6, wherein the liposomes contain about 200 nM citric acid anhydrous.

[0022] Item 8. The combination for use of any one of items 1 to 7, wherein the liposomes’ lipid bilayer comprises at least one phospholipid as main constituent.

[0023] Item 9. The combination for use of item 8, wherein the at least one phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), preferably in a range of 60 mol% to 90 mol%.

[0024] Item 10. The combination for use of any one of items 1 to 9, wherein the liposomes’ lipid bilayer comprises cholesterol, preferably in a range of 10 to 40 mol%.

[0025] Item 1 1. The combination for use of item 10, wherein the liposomes’ lipid bilayer further comprises 1 ,2-distearoyl-sn- glycero-3-phosphoethanol-amine-N-[methoxy(PEG)-2000] (DSPE-PEG), preferably in a range of 0.2 to 5 mol%.

[0026] Item 12. The combination for use of any one of items 1 to 1 1 , wherein the bilayer of the liposomes contains dipalmitoylphosphatidylcholine (DPPC), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N- [methoxy(PEG)-2000] (DSPE-PEG) at 85.5: 14:0.5 mol%, and the liposomes’ inner compartment contains citric acid anhydrous.

[0027] Item 13. The combination for use of any one of items 1 to 12, wherein the liposomes have an average diameter between about 8 pm and 15 pm (e.g., average diameter between about 10 pm and 15 pm). Item 14. The combination for use of any one of items 1 to 13, wherein the liposomal suspension contains (i) xylitol, (ii) sodium chloride, (iii) sodium hydroxide, (iv) potassium chloride, (v) calcium chloride or (vii) any combination of at least two of (i) to (v), preferably the combination comprises all of (i) to (v).

[0028] Item 15. The combination for use of any one of items 1 to 14, wherein the subject is a human, preferably a subject having ACLF.

[0029] DEFINITIONS

[0030] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0031] The terms "comprising", "having", "including", and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

[0032] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.

[0033] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0034] The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed.

[0035] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0036] Herein, the term "about" has its ordinary meaning. In embodiments, it may mean plus or minus 10% of the numerical value qualified.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0038] Liposomes

[0039] Liposomes according to the present disclosure comprise a lipid bilayer membrane.

[0040] Liposomes composition

[0041] Liposomes composition (e.g., suspension) according to the present disclosure comprise a lipid bilayer membrane enclosing a hydration medium such as an acidic buffer (acidic solution). The term “liposomal suspension” as used herein refers to a liposome composition comprising liposomes of the present disclosure (e.g., transmembrane pH- gradient liposomes of the present disclosure) suspended in an aqueous solution (e.g., neutralizing aqueous solution) of the present disclosure.

[0042] Lipid bilayer membrane

[0043] In preferred embodiments, the liposome lipid bilayer membrane comprises at least one natural or synthetic phospholipid. Preferred phospholipids are long saturated phospholipids, e.g., those having alkyl chains of more than 12, preferably more than 14, more preferably more than 16, and most preferably more than 18 carbon atoms.

[0044] In specific embodiments, the natural or synthetic phospholipid comprises at least one of 1 ,2-Dilauroyl-sn-Glycero-3- Phosphocholine (DLPC); 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC); 1 ,2-Dipalmitoyl-sn-Glycero-3- Phosphocholine (DPPC); 1 ,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC); 1 ,2-Dioleoyl-sn-Glycero-3- Phosphocholine (DOPC); 1 ,2-Dimyristoyl-sn-Glycero-3-Phosphoelhanolamine (DMPE); 1 ,2-Dipalmitoyl-sn-Glycero-3- Phosphoelhanolamine (DPPE); 1,2-Distearoyl-sn-Glycero-3-Phosphoelhanolamine (DSPE); 1 ,2-Dioleoyl-sn-Glycero- 3-Phosphoelhanolamine (DOPE); 1 -Myristoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine (MPPC); 1-Palmitoyl-2- Myristoyl-sn-Glycero-3-Phosphocholine (PMPC); 1 -Stearoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine (SPPC); 1- Palmitoyl-2-Stearoyl-sn-Glycero-3-Phosphocholine (PSPC); 1,2-Dimyristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DMPG); 1 ,2-Dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DPPG); 1,2-Distearoyl-sn-Glycero-3-[Phospho-rac- (1 -glycerol)] (DSPG); 1 ,2-Dioleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DOPG); 1 ,2-Dimyristoyl-sn-Glycero-3- Phosphate (DMPA); 1 ,2-Dipalmitoyl-sn-Glycero-3-Phosphate (DPPA); 1 ,2-Dipalmitoyl-sn-Glycero-3-[Phospho-L- Serine] (DPPS); natural L-a-phosphatidylcholine (from chicken egg, EPC, or from soy, SPC). In specific embodiments, the natural or synthetic phospholipid is DPPC. In specific embodiments, the main constituent of the liposome lipid bilayer is the at least one natural or synthetic phospholipid. In specific embodiments, the at least one natural or synthetic phospholipid forms at least 60 mol%, 65 mol%, 70 mol%, 75 mol%, 80 mol%, or 85 mol% of the liposome bilayer membrane. In specific embodiments, the natural or synthetic phospholipid forms about 85.5 mol% of the liposome bilayer membrane.

[0045] In other embodiments, the liposome lipid bilayer membrane further comprises an ammonia retention-enhancing compound. In specific embodiments, the ammonia retention-enhancing compound comprises a sterol derivative. In other specific embodiments, the sterol derivative is cholesterol. In specific embodiments, the at least one ammonia retention-enhancing compound forms at least 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 1 1 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, or 50 mol% of the liposome bilayer membrane. In specific embodiments, the at least one ammonia retention-enhancing compound forms at least 10 mol% of the liposome bilayer membrane. In specific embodiments, the at least one ammonia retention-enhancing compound forms about 14% of the liposome bilayer membrane.

[0046] In other embodiments, the liposome lipid bilayer membrane further comprises at least one steric stabilizer, such as at least one PEGylated compound, preferably at least one PEGylated lipid, more preferably DSPE-PEG. In specific embodiments, the at least one steric stabilizer forms at least 0.1 mol%, 0.2 mol%, 0.3 mol%, 0.4 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.8 mol%, 0.9 mol%, 1 mol%, 1.5 mol%, 2 mol%, 2.5 mol%, 3 mol%, 3.5 mol%, 4 mol%, 4.5 mol%, 5 mol%, 5.5 mol%, 6 mol%, 6.5 mol%, 7 mol%, 7.5 mol%, 8 mol%, 8.5 mol%, 9 mol%, 9.5 mol%, or 10 mol% of the liposome bilayer membrane. In specific embodiments, the at least one steric stabilizer forms about 0.5% of the liposome bilayer membrane.

[0047] In other embodiments, the liposome lipid bilayer membrane comprises 10 to 100 mol%, more preferably 25 to 75 mol%, more preferably 40 to 70 mol%, most preferably 50 to 60 mol% of at least one sphingolipid, preferably sphingomyelin.

[0048] In other embodiments, the liposome lipid bilayer membrane comprises 30 to 100 mol%, more preferably 40 to 95 mol%, most preferably 45 to 60 mol% of at least one surfactant. In specific embodiments, the at least one surfactant comprises hydrophobic alkyl ether (e.g., Brij ™), alkyl ester, polysorbate, sorbitan ester, and / or alkyl amide.

[0049] In other embodiments, the average diameter size of the liposomes is larger than 900 nm, larger than 1000 nm, larger than 2000 nm, larger than 3000 nm; larger than 4000 nm; larger than 5000 nm, larger than 6000 nm; larger than 7000 nm; between 3000 nm and 15 pm, between 4000 nm and 15 pm, between 5000 nm and 15 pm, between 6000 nm and 15 pm, between 7000 nm and 15 pm, between 8000 nm and 15 pm, between 3000 nm and 14 pm, between 4000 nm and 14 pm, between 5000 nm and 14 pm, between 6000 nm and 14 pm, between 7000 nm and 14 pm, between 8000 nm and 14 pm, between 3000 nm and 13 pm, between 4000 nm and 13 pm, between 5000 nm and 13 pm, between 6000 nm and 13 pm, between 7000 nm and 13 pm, or between 8000 nm and 13 pm, to avoid too rapid drainage from the peritoneal space. In specific embodiments, the average diameter size of the liposomes is between about 8 m and about 12 pm.

[0050] Hydration medium

[0051] Liposomes of the present disclosure contain a hydration medium in the inner compartment of the liposomes. In specific embodiments, the aqueous medium is an acidic buffer.

[0052] Acidic buffer / acidic solution

[0053] The acidic buffer in the inner compartment of the liposomes preferably has a high buffering capacity at low pH for a high retention of basic compounds (e.g., ammonia). The acid is not toxic to animals and does not (or only weakly) permeate out of the liposome membrane. Without being so limited, the acid enclosed in the liposomes core is (i) a hydroxy acid such as citric acid, isocitric acid, malic acid, tartaric acid, or lactic acid; (ii) a small chain fatty acid such as acetic acid; (iii) a sugar acid such as uronic acid; (iv) a dicarboxylic acid such as malonic acid; (v) a tricarboxylic acid such as propane-1 , 2, 3-tricarboxylic acid or aconitic acid; (vi) a tetracarboxylic acid such as 1 ,2,3,4-butanetetracarboxylic acid; (vii) a pentacarboxylic acid such as 1 ,2,3,4,5-pentanepentacarboxylic acid; (viii) a polymeric poly (carboxy lie acid) such as poly(acrylic acid) or poly(methacrylic acid); (ix) a polyaminocarboxylic acid such as ethylenediaminetetraacetic acid; or (x) a combination of at least two thereof. In specific embodiments, the acid is a hydroxy acid such as citric acid (e.g., citric acid anhydrous).

[0054] In specific embodiments, the concentration of acid used in a liposome preparation method such as the osmotic shock method, may be varied between 50 and 1000 mM. When a hydroxy acid such as citric acid is used, a citric acid solution of between about 100 mM and 900 mM or between about 100 mM and 900 mM, or between about 300 mM and 800 mM, or between about 400 mM and 750 mM, or between about 500 mM and 750 mM, or between about 500 mM and 650 mM or about 600 mM is optimally used; at an osmolality between 500 and 1500 mOsmol / kg, or between 600 and 1400 mOsmol / kg, or between 700 and 1400 mOsmol / kg, between 800 and 1400 mOsmol / kg, or between 800 and 1350 mOsmol / kg, or between 900 and 1350 mOsmol / kg, or between 950 and 1300 mOsmol / kg, or between 950 and 1250 mOsmol / kg, or between 1000 and 1200 mOsmol / kg is optimally used. In another specific embodiment, the concentration of citric acid (e.g., anhydrous) used in the method may be varied between 50 and 1000 mM. When a hydroxy acid such as citric acid is used, a citric acid solution of between about 600 mM is used with an osmolality of between 1000 and 1200 mOsmol / kg is used in the osmotic shock method. In a preferred embodiment, transmembrane pH-gradient liposomes produced by methods described herein have an inner concentration of citric acid anhydrous of about 200 nM, and an inner osmolarity that is physiological i.e. , around 350 mOsmol / kg.

[0055] The acid within the core (inner compartment of liposomes) is present in a concentration that produces a pH between 1 and 6 in the core of the liposomes, and in a specific embodiment, a pH between 1.5 and 3, and in a more specific embodiment, a pH of about 2 in the core of the liposomes.

[0056] In a specific embodiment, the liposomes contain in their internal compartment / core between 200 nM citric acid (anhydrous), and this core has a pH of about 2.

[0057] In alternative embodiments, liposomes for use in the present disclosure are as described in EP 2 882 421 to Leroux et al. the content of which is herein incorporated by reference.

[0058] Composition

[0059] In accordance with another aspect of the present disclosure, there is provided a composition (in the form of a suspension or otherwise) comprising the liposomes of the present disclosure, and at least one pharmaceutically acceptable excipient or carrier. The compositions of the disclosure can contain a pharmaceutically acceptable carrier / excipient including, without limitation, aqueous or non-aqueous solutions. Pharmaceutically acceptable carriers also can include physiologically acceptable aqueous vehicles (e.g., sugar solutions, saline), neutralizing species (basic or acidic, such as weak bases or weak acids) but also chemical agents used to adjust the osmolarity and / or provide a physiological function. Without being limited excipients encompassed by the present disclosure include glycerol, tris((hydroxymethyl)aminomethane) (TRIS), agents to counteract potential anticoagulant effects of certain weak acids (e.g., citric acid) such as calcium salts (e.g., calcium chloride); other salts such as sodium salts (e.g., sodium chloride), magnesium salts, lactate salts, potassium salts (e.g., potassium chloride); hydroxides (e.g., sodium hydroxide); sugars or polysaccharides (e.g., icodextrin, glucose, sorbitol, fructose); amino acids; sugar alcohols (e.g., xylitol, glycerol) or other known carriers / excipients appropriate for the intraperitoneal route. In specific embodiments, the liposomal composition (e.g., suspension) comprise (I) xylitol, (II) sodium chloride, (ill) sodium hydroxide, (iv) potassium chloride, (v) calcium chloride or (vii) any combination of at least two of (I) to (v), preferably the combination comprises all of (I) to (v).

[0060] Method of preparing liposomes

[0061] Osmotic shock method

[0062] In specific embodiments, a lipid blend can be prepared by mixing the lipid bilayer components in a solvent such as an alcohol or a mixture of water and of an organic solvent (e.g., alcohol such as ethanol or t-butanol), until complete dissolution to form a homogenous lipid mix. The mix can be conducted at room temperature (i.e., around 20-25 °C) or while heating (e.g., at a temperature of up to 60 °C, preferably up to 45 °C) and optionally slowly mixing.

[0063] The mix can optionally be filtered (e.g., 0.2 pm filter). The organic solvent is then removed e.g., by lyophilization, spray drying (e.g., using liquid nitrogen as drying gas), rotary evaporation or otherwise.

[0064] The resulting dried lipid blend can then be hydrated in the aqueous medium as further described below.

[0065] Aqueous medium

[0066] In a preferred embodiment, the lipid bilayer components can be directly mixed in an aqueous medium having an osmolarity of not more than 400 mOsmol / l (direct lipid hydration method).

[0067] In an embodiment, the aqueous medium has a pH value of around 7, e.g., in the range of 6.0 to 7.5, of 6.1 to 7.4, of 6.2 to 7.3, of 6.3 to 7.2, of 6.4 to 7.1 , of 6.5 to 7.3, of 6.6 to 7.3, of 6.7 to 7.3, of 6.8 to 7.3, of 6.9 to 7.1 , of 6.95 to 7.01 , or of about 7.0. In an embodiment, the aqueous medium is chosen from the group consisting of water (e.g., distilled water, deionized water, ultra-pure water or any other kind of purified water), a mixture of water as defined above and of an organic solvent (e.g., alcohol), aqueous solutions of organic salts, aqueous solutions of inorganic salts, aqueous solutions of organic substances, and combinations thereof. In an embodiment, the aqueous medium is chosen from the group consisting of aqueous solutions of organic salts having a pH value of around 7, aqueous solutions of inorganic salts having a pH value of around 7, aqueous solutions of organic substances having a pH value of around 7, water and combinations thereof.

[0068] When using organic or inorganic salts or other organic compounds, these salts or compounds are present in the aqueous medium, in an embodiment, in a low concentration so as to keep a difference in osmolarity between the aqueous medium and the acidic or basic (preferably acidic) hyperosmotic buffer provoking the osmotic shock, which difference is large enough to induce the diffusion of the acidic or basic (preferably acidic) hyperosmotic buffer into the vesicle internal compartment.

[0069] The aqueous medium is a medium that resembles water (in particular with respect to pH) but that might contain a low concentration of salts or compounds, e.g., for buffering the pH value in a neutral range.

[0070] As indicated above, the aqueous medium has an osmolarity of not more than 400 mOsm / l. In an embodiment, the osmolarity of the aqueous medium is equal to or less than 300 mOsm / l, equal to or less than 250 mOsm / l, equal to or less than 200 mOsm / l, equal to or less than 150 mOsm / l, equal to or less than 100 mOsm / l, equal to or less than 75 mOsm / l, equal to or less than 50 mOsm / l, equal to or less than 25 mOsm / l, equal to or less than 10 mOsm / l, equal to or less than 5 mOsm / l equal to or less than 1 mOsm / l. In an embodiment, the osmolarity is in the range of 1 mOsm / l to 200 mOsm / l or in the range built up from any of the before mentioned osmolarities (such as 10 mOsm / l to 150 mOsm / l etc.). In an embodiment, the osmolarity of the aqueous medium is in a range between 0 mOsm / l and 49 mOsm / l, between 0 mOsm / l and 45 mOsm / l, or between 0 mOsm / l and 40 mOsm / l; in particular between 0 mOsm / l and 35 mOsm / l; more particularly between 0 mOsm / l and 30 mOsm / l, or between 0 mOsm / l and 25 mOsm / l.

[0071] In specific embodiments, the liposomes can optionally be extruded or filtered to obtain liposomes having a specific size.

[0072] The hydration of the lipid bilayer components / lipid blend can be conducted at room temperature (i.e., around 20-25 °C) or while heating (e.g., at a temperature of up to 60 °C (e.g., prewarmed aqueous medium), preferably up to 45 °C) and optionally slowly stirring for a period of about 15 minutes to 4 hours, preferably about 2 hours. At that stage, the final concentration of lipids is preferably at about 100 mg / g, if the hydration was performed while heating, the mixture is cooled down to room temperature (i.e., around 20-25 °C). The mixture can optionally be degassed (e.g., under vacuum) to remove air bubbles.

[0073] In an embodiment, the hydrated liposomes so prepared are sterilized so as to obtain sterilized liposomes or sterilized suspension containing the liposomes. The sterilization can be carried out by, e.g., sterile filtration or steam sterilized (e.g., autoclaving), e.g., for a period of about 5 minutes to 2 hours, 10 minutes to 1 hour, or about 15 minutes, or about 30 minutes.

[0074] In another embodiment, the vesicles are stored for a first period of time prior to carrying out the step of mixing the liposomes (or the liposomes-containing suspension) with the acidic buffer. This storage can be optimally accomplished if the liposomes are sterilized after the hydration in aqueous medium step because then, no or little degradation processes will occur in the sterilized liposomes suspension. The first period of time can be one day, a few days (e.g., 1, 2, 3 or 4 days), one week, several weeks (e.g., 1 , 2, 3 or 4 weeks), one month or even several months (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 months). Sterilized liposomes contained in an aqueous medium are stable entities. Since they do not yet contain any specific basic buffer used to prepare the pH gradient, no buffer loss due to liposomes bilayer degradation or leakage of the liposomes has to be feared. This is also true if the liposomes, in an embodiment, contain low amounts of electrolytes molecules since an according osmolarity within the vesicles would then be in a range of between 0 or 1 mOsm / l to 200 mOsm / l.

[0075] Acidic and hyperosmotic buffer

[0076] Thereafter, the hydrated (and optionally sterilized) liposomes are mixed with an acidic buffer having an osmolarity being at least 200 mOsm / l higher than the osmolarity of the aqueous medium to apply an osmotic shock to the liposomes and to obtain buffer-filled liposomes. In an embodiment, the osmolarity of the acidic buffer is at least 220 mOsm / l higher than the osmolarity of the aqueous medium, at least 250 mOsm / l higher, at least 300 mOsm / l higher, at least 350 mOsm / l higher, at least 400 mOsm / l higher, at least 450 mOsm / l higher, at least 500 mOsm / l higher, at least 550 mOsm / l higher, at least 600 mOsm / l higher, at least 650 mOsm / l higher, at least 700 mOsm / l higher, at least 750 mOsm / l higher, at least 800 mOsm / l higher, at least 850 mOsm / l higher, at least 900 mOsm / l higher, at least 950 mOsm / l higher, at least 1000 mOsm / l higher, at least 1050 mOsm / l higher, at least 1100 mOsm / l higher or at least 1200 mOsm / l higher. In an embodiment, the osmolarity of the acidic buffer is in a range of 200 mOsm / l to

[0077] 1100 mOsm / l higher than the osmolarity of the aqueous medium or in a range built up from any of the before mentioned osmolarities (such as 220 mOsm / l to 1200 mOsm / l etc.).

[0078] Thus, the acidic buffer is a hyperosmotic buffer with respect to the aqueous medium used in the liposome’s hydration step. In doing so, an osmotic shock is extemporaneously applied to the liposomes. This osmotic shock results in incorporating the acidic buffer within the liposomes. Thus, the osmotic shock serves for a short-term destabilization of the liposomes in order to allow buffer incorporation into the liposomes. Buffer-filled liposomes result. In an embodiment, the hyperosmotic buffer can also contain electrolytes that are used to modulate the osmolarity or have a physiological function.

[0079] A sufficient amount of the acidic buffer is to be added to the liposomes suspended in the aqueous medium since otherwise no osmotic shock will be achieved. A sufficient amount can be, depending on the difference between the osmolarity of the aqueous medium and the osmolarity of the basic buffer, a volume that corresponds to at least 0.1 times the volume of the aqueous medium, at least 0.3 times, at least 0.5 times, at least 0.8 times, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times or at least 5 times. In an embodiment, the acidic buffer can be added in a volume that equals the volume of the aqueous medium. In an embodiment, the volume of the acidic buffer to be added can be 0.1 times to 5 times the volume of the aqueous liposome suspension or any other range that can be built up from the above-mentioned values (such as 0.3 times to 3 times, etc.). In an embodiment, the pH value of the hyperosmotic buffer is in a range of pH 1 to pH 6.9, pH 1 .5 to pH 6.5, pH 1 .5 to pH 6.0, pH 1 .5 to pH 5.5, pH 1 .5 to pH 5.0, pH 1 .5 to pH 4.5, pH 1 .5 to pH 4.0, pH 1 .5 to pH 3.5, pH 1 .5 to pH 3.0, pH 1 .5 to pH 2.5, pH 1 .5 to pH 2.0, pH 2.0 to pH 6.0, pH 2 to pH 5.5, pH 2.0 to pH 5.0, pH 2.0 to pH 4.5, or pH 2.0 to pH

[0080] 3.5.

[0081] In specific embodiments, the hyperosmotic buffer can contain additional chemical agents such as a complexing agent or chelating agent.

[0082] In specific embodiments, the hyperosmotic buffer comprises salts such as but not limited to sodium chloride, sodium hydroxide, and / or magnesium chloride.

[0083] In specific embodiments where a sterile transmembrane pH gradient liposome is preferred, the acidic buffer is sterilized. In such embodiments, where the hydrated liposomes had also been sterilized before loading the acidic buffer, fully sterile buffer-filled liposomes or a fully sterile suspension containing buffer-filled liposomes are prepared. The sterilization can be carried out by, e.g., sterile filtration or autoclaving.

[0084] In an embodiment, the mixture of the aqueous medium and the basic or acidic buffer in which the buffer-filled liposomes are suspended has an osmolarity of at least 200 mOsm / l, of at least 220 mOsm / l, of at least 250 mOsm / l, of at least 300 mOsm / l, of at least 350 mOsm / l, of at least 400 mOsm / l, of at least 450 mOsm / l, of at least 500 mOsm / l, or of at least 550 mOsm / l. In an embodiment, the osmolarity is in the range of 200 mOsm / l to 550 mOsm / l or in the range built up from any of the before mentioned osmolarities (such as 220 mOsm / l to 500 mOsm / l etc.).

[0085] The liposomes acidic buffer mixture can optionally be incubated. In specific embodiments, the mixture is stirred (e.g., by orbital shaking) at e.g., room temperature (i.e., around 20-25 °C).

[0086] Neutralizing aqueous solution

[0087] Then, a mixture of the aqueous medium and the acidic buffer containing the buffer-filled liposomes is diluted by adding a neutralizing aqueous solution. The mixture of acidic buffer and neutralizing solution makes up a suspension buffer. Thus, after dilution, transmembrane pH-gradient liposomes suspended in the suspension buffer result. Thereby, the pH of the suspension buffer differs from the acidic buffer contained in the buffer-filled liposomes. The pH difference is in an embodiment at least 1 pH unit, at least 1.5 pH units, at least 2 pH units, at least 2.5 pH units, at least 3 pH units, at least 3.5 pH units, at least 4 pH units, at least 4.5 pH units, at least 5 pH units, at least 5.5 pH units, at least 6 pH units, at least 6.5 pH units, or at least 7 pH units.

[0088] In an embodiment, the pH value of the neutralizing solution is in a range of pH 7.1 to pH 14, pH 7.1 to pH 13.5, pH 7.1 to pH 13.0, pH 7.1 to pH 12.5, pH 7.1 to pH 12, pH 7.1 to pH 11 .5, pH 7.1 to pH 11 .0, pH 7.1 to pH 10.5, pH 7.1 to pH 10, pH 7.1 to pH 9.5, pH 7.1 to pH 9.0, pH 7.1 to pH 8.5, pH 7.2 to pH 14, pH 7.2 to pH 13.5, pH 7.2 to pH 13, pH 7.2 to pH 12.5, pH 7.2 to pH 12, pH 7.2 to pH 1 1.5, pH 7.2 to pH 1 1 , pH 7.2 to pH 10.5, pH 7.2 to pH 10, pH 7.2 to pH

[0089] 9.5, pH 7.2 to pH 9, pH 7.2 to pH 8.5, pH 7.3 to pH 14, pH 7.3 to pH 13.5, pH 7.3 to pH 13, pH 7.3 to pH 12.5, pH 7.3 to pH 12, pH 7.3 to pH 1 1.5, pH 7.3 to pH 1 1 , pH 7.3 to pH 10.5, pH 7.3 to pH 10, pH 7.3 to pH 9.5, pH 7.3 to pH 9, pH 7.3 to pH 8.5, pH 7.4 to pH 14, pH 7.4 to pH 13.5, pH 7.4 to pH 13, pH 7.4 to pH 12.5, pH 7.4 to pH 12, pH 7.4 to pH 11.5, pH 7.4 to pH 1 1 , pH 7.4 to pH 10.5, pH 7.4 to pH 10, pH 7.4 to pH 9.5, pH 7.4 to pH 9, pH 7.4 to pH 8.5, pH 7.5 to pH 14, pH 7.5 to pH 13.5, pH 7.5 to pH 13, pH 7.5 to pH 12.5, pH 7.5 to pH 12, pH 7.5 to pH 1 1.5, pH 7.5 to pH 1 1 , pH 7.5 to pH 10.5, pH 7.5 to pH 10, pH 7.5 to pH 9.5, pH 7.5 to pH 9, pH 7.5 to pH 8.5, pH 8.0 to pH 13.0, pH 8.5 to pH 12.5, pH 9.0 to pH 13, pH 9.0 to pH 12.5, pH 9.0 to pH 12.0, pH 9.5 to pH 11.5, pH 10 to pH 13, pH 10 to pH 12.5, pH 10 to pH 12.0, pH 10 to pH 1 1.5, pH 10 to pH 11 , pH 10 to pH 12.5, pH 10.5 to pH 12.0, pH 10.5 to pH 13, pH 10.5 to pH 12.5, pH 10.5 to pH 12.0, pH 10.5 to pH 1 1.5, or pH 10.5 to pH 1 1. In a specific embodiment, the pH of the neutralizing solution is about 12.5.

[0090] In an embodiment, the neutralizing solution has a composition designed to avoid disrupting the buffer filled vesicles so as to not destabilize these vesicles. It may contain neutralizing species (basic or acidic, such as weak bases or weak acids) but also chemical agents used to adjust the osmolarity and / or provide a physiological function. Calcium salts can be added in the preparation process to counteract the anticoagulant effects of some weak acids (e.g., citric acid). This is of particular importance if the vesicles are to be used in in vivo applications. Sodium hydroxide, sodium salts (such as sodium chloride), potassium chloride, calcium chloride magnesium salts, lactate salts, glycerol, icodextrin, glucose, sorbitol, fructose, amino acids or xylitol can also be used as ingredients of the neutralizing solution. In specific embodiments, the neutralizing solution contains

[0091] In an embodiment, the neutralizing solution has an osmolarity of between 250 mOsm / l and 550 mOsm / l, of between 270 and 520 mOsm / l, of between 290 and 500 mOsm / l, of between 300 and 480 mOsm / l, of between 320 and 450 mOsm / l, of between 330 and 420 mOsm / l, of between 350 and 400 mOsm / l, of between 375 and 400 mOsm / l, of between 385 and 400 mOsm / l or of between 390 and 400 mOsm / l.

[0092] In an embodiment, the neutralizing solution has an osmolarity which is less than 200 mOsm / l higher or lower than the osmolarity of the mixture containing the buffer-containing vesicles (i.e., the buffer-containing vesicles solution), in particular less than 150 mOsm / l higher or lower, in particular less than 100 mOsm / l higher or lower, in particular less than 50 mOsm / l higher or lower, in particular less than 20 mOsm / l higher or lower, or in particular less than 10 mOsm / l higher or lower. In an embodiment, the difference in osmolarity between the neutralizing solution and the mixture containing the buffer-containing vesicles is between 1 mOsm / to 200 mOsm / l, in particular between 10 mOsm / to 150 mOsm / l, in particular between 20 mOsm / to 100 mOsm / l, in particular between 30 mOsm / to 80 mOsm / l, pr in particular between 40 mOsm / to 60 mOsm / l.

[0093] Due to the pH differences between the suspension buffer and the acidic buffer, a transmembrane pH-gradient between the inner part of the liposomes and the surrounding suspension buffer is achieved. The resulting transmembrane pH-gradient can be used in accordance with the present disclosure.

[0094] In an embodiment, the pH value of the suspension buffer containing the transmembrane pH-gradient vesicles is in the range of 5.5 to 8.5, of 6.0 to 8.0, of 6.3 to 7.7, of 6.3 to 7.5, of 6.3 to 7.3, of 6.3 to 7.2, of 6.3 to 7.1 , of 6.5 to 7.7, of 6.5 to 7.5, of 6.5 to 7.3, of 6.5 to 7.2, of 6.5 to 7.1 , of 6.8 to 7.5, or of 7.0 to 7.4. Thus, the suspension buffer may have a physiological pH value. In a specific embodiment, the pH value of the suspension buffer is about 6.5.

[0095] Osmotic shock methods are also described in EP 3 291 797 to Leroux et al. the content of which is herein incorporated by reference.

[0096] Alternative methods of preparing liposomes

[0097] In another embodiment, the method of preparing the liposomes (e.g., transmembrane pH-gradient liposomes) includes the film hydration method. For example, liposomes bilayer membrane components are dissolved in an organic solvent (e.g., dichloromethane: methanol), the organic solvent is then removed (e.g., by rotary evaporation) to form a dried lipid film. The dried lipid can be stored for future use (e.g., under vacuum). The dried lipid can thereafter be hydrated directly in the buffer such as the acidic buffer described above, and the external solution exchanged with a neutral solution as described above. Alternatively, the film hydration method can first be used to form a lipid film which is then hydrated in the aqueous medium as described above.

[0098] The aqueous medium loaded liposomes can thereafter be subjected to the osmotic shock step described above to load the acidic buffer therein and be subjected to the neutralization solution step described above to create the transmembrane pH-gradient liposomes suspension.

[0099] Alternatives of methods of preparing liposomes are also described in EP 2 882 421 to Leroux et al. the content of which is herein incorporated by reference.

[0100] Alternatively, the liposomes can be prepared by spray drying as described herein.

[0101] Albumin

[0102] Human albumin is the most abundant globular plasma protein (3.5 - 5.0 g / dl), with a molecular weight of 67 kDa. It is an ampholyte molecule, which has a negative surface charge and is water soluble. Albumin is synthesized by the hepatocytes in the liver. Albumin has 7 binding sites, from which Sudlow sites I and II are the main binding sites. While the present disclosure uses human serum albumin (Recombinant human albumin, lyophilized powder, low endotoxin, suitable for cell culture, Sigma-Aldrich, SAE0160-5G, Source: SLCQ8521) in experiments described herein, it is understood that any form of albumin commonly used in clinical settings is encompassed by the present disclosure. More particularly, any albumin appropriate for use in albumin dialysis may be used in accordance with the present disclosure, including human serum albumin (HSA) such as human mercaptalbumin (reduced form of albumin which has a free thiol group at Cys34), recombinant human albumin, and fragments thereof comprising bile acid binding sites.

[0103] Route of administration and mechanism of action The liposomes of the present disclosure are intraperitoneally administered. The albumin of the present disclosure is intraperitoneally administered or intravenously administered.

[0104] The term “intraperitoneal administration” as used herein is meant to be understood as it is commonly understood by the person skill in the art of peritoneal dialysis treatment. For practicing the disclosure, a pharmaceutically effective amount of the liposome (e.g., transmembrane pH-gradient liposome) suspension of the disclosure (e.g., VS-01) is administered into the peritoneal cavity, e.g., by injection as a single bolus, by continuous infusion or by perfusion, e.g., by catheter, such as a catheter commonly used for paracentesis.

[0105] The transmembrane pH-gradient liposomes within the cavity and the nearby tissues and organs take up the circulating (blood) bile acid(s) (particularly cholic acid, glycocholic acid and / or glycochenodeoxycholic acid) and reduce the concentration thereof in blood. The transmembrane pH-gradient liposomes within the cavity and the nearby tissues and organs will also take up the circulating (blood) ammonia based on the pH gradient across the liposome membrane. The acidic buffer contained within the liposomes possesses a lower pH than the physiological pH in the peritoneal cavity (which is about 7.5 to 8). Hence, ammonia can diffuse through the hydrophobic liposome bilayer in its uncharged state and be then trapped in its protonated (ionized) state (e.g., ammonium) in the inner liposome compartment.

[0106] The liposomes (e.g., transmembrane pH-gradient liposomes) thus sequester bile acid(s) (particularly cholic acid, glycocholic acid and / or glycochenodeoxycholic acid) (and ammonia) and reduce the toxic concentration of these compound(s) within the cavity, nearby tissues and organs and blood. The liposomes in the peritoneal cavity are then removed / extracted from the peritoneal cavity with the fluid present therein (dialysate). Intraperitoneal administration and extraction can be performed subsequently (sequentially) and / or simultaneously. Without being so limited, the dialysate can be extracted by passive drainage through a catheter by gravity or pumped out by suction via a pump such as a peristaltic pump used for infusion.

[0107] Diseases

[0108] Pathological blood level of bile acids (particularly a pathological blood level of total bile acids or hydrophobic bile acids such as cholic acid, glycocholic acid and / or glycochenodeoxycholic acid)

[0109] The present disclosure provides a combination of liposomes (e.g., transmembrane pH-gradient liposomes) of the present disclosure and albumin for use in reducing a pathological blood level of bile acids (particularly a pathological blood level of total bile acids or hydrophobic bile acids such as cholic acid, glycocholic acid and / or glycochenodeoxycholic acid). It is further for use in the treatment of one or more disease(s) or disorder(s) associated with a pathological blood level of bile acids (particularly a pathological blood level of total bile acids or hydrophobic bile acids such as cholic acid, glycocholic acid and / or glycochenodeoxycholic acid) as defined herein below. In other specific embodiments, it is for use in the treatment of one or more pre-existing disease(s) or disorder(s) as defined herein below. In specific embodiments, the combination of liposomes (e.g., transmembrane pH-gradient liposomes) of the present disclosure and albumin is administered to subjects without having first measured their blood levels of bile acids (particularly their blood levels of hydrophobic bile acids such as cholic acid, glycocholic acid and / or glycochenodeoxycholic acid). For example, in specific embodiments, the combination of liposomes (e.g., transmembrane pH-gradient liposomes) of the present disclosure and albumin is administered to subjects diagnosed with one or more pre-existing disease(s) or disorder(s) as defined herein below, without having first measured their blood levels of bile acids (particularly their blood levels of hydrophobic bile acids such as cholic acid, glycocholic acid and / or glycochenodeoxycholic acid).

[0110] Without being so limited, a pathological blood level of total bile acids as used herein is meant to refer to > 2-10 pimol / L A pathological blood level of fasting total bile acids as used herein is meant to refer to >1 .5-3.1 pimol / L A pathological blood level of cholic acid as used herein is meant to refer to > 0.01-0.05 pimol / L; a pathological blood level of glycocholic acid as used herein is meant to refer to > 0.03-0.1 pimol / L; and a pathological blood level of glycochenodeoxycholic acid as used herein is meant to refer to >0.05-0.36 pimol / L

[0111] Disease(s) or disorder(s) associated with a pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid)

[0112] As used herein, the term “disease(s) or disorder(s) associated with a pathological blood level of hydrophobic bile acids” refers to e.g., disease(s) or disorder(s) caused by a pathological blood level of hydrophobic bile acids.

[0113] Without being so limited, it refers to at least one of a pathological blood level of hydrophobic bile acids-induced liver impairment, cholestasis, jaundice, gallstones, digestive impairment, metabolic disorder, or neurological impairment. As used herein the term “pathological blood level of hydrophobic bile acid-induced” is used to denote that the disease or disorder is mainly caused by a pathological blood level of hydrophobic bile acid rather than from another (known or unknown, preferably known) underlying disease or disorder in the subject.

[0114] Pre-existing disease or disorder

[0115] As used herein, the term “pre-existing disease or disorder” refers to any disease or disorder that the subject has prior to the onset of a pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid) or before receiving a diagnosis of this pathology. In specific embodiments, the pre-existing disease or disorder contributes to a pathological blood level of hydrophobic bile acids. For example, the subject has a pre-existing disease or disorder if she / he received a diagnosis of another disease or disorder at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, etc. prior to receiving a diagnosis of a pathological blood level of hydrophobic bile acids. In specific embodiments, the pre-existing disease or disorder is hypercholanemia, pre-existing ascites, pre-existing hyperammonemia, pre-existing liver impairment or pre-existing kidney impairment. In a specific embodiment, the pre-existing liver impairment is compensated or decompensated cirrhosis, preferably decompensated. In another specific embodiment, the pre-existing liver impairment is acute-on-chronic liver failure (ACLF). In another specific embodiment, the pre-existing kidney impairment is kidney failure.

[0116] Bile acid levels Table I: Comparison of serum levels of bile acids in acute decompensation (AD) versus no AD patients as well as AD and acute-on-chronic liver failure (ACLF) patients. It shows that mainly primary bile acids are increased in AD and ACLF.

[0117] Cholic acid = up

[0118] Taurocholic acid up up

[0119] Glycocholic acid up up

[0120] Taurochenodeoxycholic acid up up

[0121] Glycochenodeoxycholic acid up up

[0122] Chenodeoxycholic acid up up

[0123] Lithocholic acid =

[0124] Taurolithocholic acid =

[0125] Glycol ithocholic acid =

[0126] Deoxycholic acid down

[0127] Taurodeoxycholic acid =

[0128] Glycodeoxycholic acid =

[0129] Tertiary bile acids

[0130] Ursodeoxycholic acid =

[0131] Tauroursodeoxycholic acid = Glycoursodeoxycholic acid up

[0132] Subjects

[0133] As used herein the terms “subject” or “subject in need thereof” refer to a subject who has a pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid). In specific embodiments, the subject has a disease or disorder associated with a pathological blood level of hydrophobic bile acids (particularly cholic acid, glycocholic acid and / or glycochenodeoxycholic acid) (e.g., hyperammonemia, liver impairment and / or kidney impairment). In specific embodiments, the subject further has a pre-existing disease or disorder. In other specific embodiments, the subject does not have a pre-existing disease or disorder. In other specific embodiments, the subject has pre-existing ascites. In other specific embodiments, the subject has pre-existing hyperammonemia. In other specific embodiments, the subject has pre-existing liver impairment (e.g., cirrhosis or ACLF). In other specific embodiments, the subject has preexisting kidney impairment. In other specific embodiments, the subject has two of, or three of or all four of pre-existing ascites, pre-existing hyperammonemia, pre-existing liver impairment and pre-existing kidney impairment. In other specific embodiments, the subject does not have pre-existing ascites. In other specific embodiments, the subject does not have pre-existing hyperammonemia. In other specific embodiments, the subject does not have pre-existing liver impairment (e.g., cirrhosis or ACLF). In other specific embodiments, the subject does not have pre-existing kidney impairment. In other specific embodiments, the subject does not have two of, or three of or all four of pre-existing ascites, pre-existing hyperammonemia, pre-existing liver impairment and pre-existing kidney impairment. The term subject refers to an animal, to a mammal or more specifically to a human in a specific embodiment. The liposomes or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) of the present disclosure may also be used for veterinary applications and be used for pets or other animals (e.g., pets such as cats, dogs, horses, etc.; and cattle, fishes, swine, poultry, etc.). In specific embodiments, the subject has a healthy liver and / or does not suffer from drug-induced hyperammonemia. In a specific embodiment, the subject is an adult.

[0134] In certain embodiments, the methods of the present disclosure encompass a step of diagnosing the subject.

[0135] Concentration

[0136] The specific concentration of each of the liposomal suspension (comprising transmembrane pH gradient liposomes), and the albumin in the combination of the present disclosure is selected taking into account the subject being treated and to achieve optimal effect in terms of bile acid removal. The concentration of the liposomal suspension used prior to administration to subjects is typically between 2 mM and 10 mM total lipid, and the concentration of albumin used prior to administration to subjects is typically between 6 mM and 15 mM. After intraperitoneal administration to the subject, the liposomal suspension and the albumin are further diluted by liquids from the peritoneal space. The in situ concentration of each of the liposomal suspension and of the albumin in the combination of the present disclosure is thus smaller than that administered.

[0137] Combination therapy

[0138] The present disclosure encompasses combining an intraperitoneal administration of the liposomes or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) as described herein (e.g., VS-01) with an intraperitoneal or intravenous administration of albumin with one or more further therapies for treating a pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid), a disease or disorder associated with a pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid) or a pre-existing disease or disorder (simultaneously or sequentially depending on the nature of the additional treatment).

[0139] For example, the combination can comprise the liposomes or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) of the present disclosure (e.g., VS-01) and albumin, with ursodiol (Actigall, Urso, Urso Forte) for treating the pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid).

[0140] Also, the combination therapy can include a liposome or composition or suspension thereof (e.g., transmembrane pH- gradient liposomes or composition or suspension thereof) of the present disclosure combined with at least one other drug or therapy used for the prevention or treatment of a disease or disorder associated with a pathological blood level of hydrophobic bile acids such as hyperammonemia or of a pre-existing disease or disorder such as a cirrhosis or ACLF. In this context, examples of therapeutic agents or therapies that may be administered in combination (simultaneously or sequentially) with the liposomes or composition or suspension thereof (e.g., transmembrane pH- gradient liposomes or composition or suspension thereof) of the present disclosure include another liposomes or composition or suspension thereof of the present disclosure and / or at least one other therapeutic agent or therapy. When used to treat hyperammonemia, the at least one other therapeutic agent or therapy can be at least one of nonabsorbable disaccharides such as lactulose or lactilol, rifaximin, a branched-chain amino acid, neomycin, metronidazole, probiotic (such as but not limited to VSL#3 (Rivera-Flores 2020)), a glutaminase inhibitor, L-ornithine- L-aspartate, hemodialysis, peritoneal dialysis, sodium phenylbutyrate (e.g., Buphenyl®), sodium phenylacetate, sodium benzoate, a combination of sodium phenylacetate / sodium benzoate (e.g., Ammonul®, Ucephan®), glycerol phenylbutyrate (e.g., Ravicti®) or carglumic acid. When used to treat a urinary tract infection, the at least one other therapeutic agent can be an antibiotic such as trimethoprim / sulfamethoxazole (Bactrim™, Septra™, others), fosfomycin (Monurol ™), nitrofurantoin (Macrodantin™, MacroBID™), cephalexin (Keflex™), ceftriaxone, a fluoroquinolone such as ciprofloxacin (Cipro™), levofloxacin and others. When used to treat an ulcer, the at least one other therapeutic agent can be an antibiotic such as amoxicillin (Amoxil™), clarithromycin (Biaxin™), metronidazole (Flagyl™), tinidazole (Tindamax™), tetracycline and levofloxacin; a proton pump inhibitor such as omeprazole (Prilosec™), lansoprazole (Prevacid™), rabeprazole (Aciphex™), esomeprazole (Nexium™) and pantoprazole (Protonix™); an acid blocker such as famotidine (Pepcid AC™), cimetidine (Tagamet HB™) and nizatidine (Axid AR™), an antacid that neutralize stomach acid; and / or cytoprotective agents such as sucralfate (Carafate™) and misoprostol (Cytotec™).

[0141] When used in such combination, the liposomes or composition or suspension thereof of the present disclosure could enable the administration of a lower dose of the other drug or therapy (e.g., anti-hyperammonemia drug such as lactulose) and thereby reduce the side effects associated with such drug or therapy, such as diarrhea, nausea, bloating, and flatulence.

[0142] Treatment and prevention

[0143] The present disclosure encompasses the use of the liposomes or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) and albumin as described herein for the treatment of a pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid) in a subject as further described herein.

[0144] The terms “treat / treating / treatment” as used herein, refers to eliciting the desired biological response, i.e., a therapeutic effect. In accordance with the disclosure herein, the therapeutic effect comprises one or more of a decrease / reduction in the frequency, duration and / or severity of a pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid) or of at least one symptom thereof. It may further comprise one or more of a decrease / reduction of frequency, duration and / or severity of at least one a symptom triggered by a pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid), and / or duration of symptom-free periods following administration of the combination of liposomes and albumin of the present disclosure as described herein, alone or in combination with another agent for the treatment of a pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid) or at least one symptom thereof.

[0145] The terms “prevent / preventing / prevention” as used herein, refers to eliciting the desired biological response, i.e., a prophylactic effect. In accordance with the disclosure provided herein, in some embodiments, a prophylactic effect comprises a complete or partial avoidance / inhibition of at least one symptom of a pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid) or a symptom thereof following administration of the combination of the present disclosure, alone or in combination with another agent for the prevention or treatment of a pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid) or of at least one a symptom thereof.

[0146] In some embodiments, "therapeutically effective amount" or “effective amount” or "therapeutically effective dosage" of a combination of the present disclosure results in a treatment of a pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid) in a subject in need thereof. It may also result in the treatment or prevention of at least one a symptom thereof in a subject.

[0147] As used herein the term “a symptom of a pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid)” refers to any of the clinical characteristics of subjects having a pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid) and correspond to events resulting at least in part from the pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid) per se. For example, it includes hyperammonemia, liver impairment, kidney impairment, etc.

[0148] As used herein the term “higher” in reference to the pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid) as compared to the reference levels (e.g., level ranges considered normal according to literature i.e. level ranges in corresponding (e.g., gender matched if levels vary according to sex) healthy subject). In specific embodiments it refers to an increase of at least 10% higher than the reference level; at least 15% higher; at least 20% higher; at least 25% higher; at least 30% higher; at least 35% higher; at least 40% higher; at least 45% higher; at least 50% higher; at least 55% higher; at least 65% higher; at least 70% higher; at least 75% higher; at least 80% higher; at least 85% higher; at least 90% higher; at least 95% higher; at least 100% higher; at least 1 10% higher; at least 120% higher; at least 130% higher; at least 140% higher; at least 150% higher; at least 160% higher; at least 170% higher; at least 180% higher; at least 190% higher; at least 200% higher; at least 210% higher; at least 220% higher; or at least 230% higher or more than the reference level.

[0149] As used herein the term “reduce” or “reduction” in reference to the effect of the combination of the present disclosure (liposomes or composition or suspension thereof (e.g., transmembrane pH-gradient liposomes or composition or suspension thereof) + albumin) on the pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid) in a subject in need thereof after treatment with (i.e. after being administered) the combination as compared to the corresponding level in the subject in need thereof prior to treatment. In other specific embodiments, the comparison to determine the reduction is with a corresponding reference level of untreated subjects that have a pathological blood level of hydrophobic bile acids. In specific embodiments, it refers to a reduction of the pathological blood level of hydrophobic bile acids (particularly a pathological blood level of cholic acid, glycocholic acid and / or glycochenodeoxycholic acid) of at least 10% compared to the level in the subject in need thereof prior to the treatment; a reduction of at least 15%; a reduction of at least 20%; a reduction of at least 25%; a reduction of at least 30%; a reduction of at least 35%; a reduction of at least 40%; a reduction of at least 45%; a reduction of at least 50%; a reduction of at least 55%; a reduction of at least 65%; a reduction of at least 70%; a reduction of at least 75%; a reduction of at least 80%; a reduction of at least 85%; a reduction of at least 90%; or a reduction of at least 95% or more, so as to reach the level found in a corresponding healthy subject.

[0150] Kits

[0151] The present disclosure also provides a kit comprising (a) a liposome aqueous suspension (e.g., containing (optionally sterilized) liposomes in an aqueous medium (e.g., with a pH value of around 7, e.g., in the range of 6.0 to 7.5) as described herein; (b) a hydration medium (e.g., acidic buffer) as described herein; and (c) an aqueous solution as described herein (e.g., a neutralizing aqueous solution as described herein); and optionally (d) (I) instructions to use (a) to (c) in combination with albumin to treat a pathological blood level of bile acids as described herein; (II) albumin as described herein; (ill) at least one other drug for the prevention or treatment of a pathological blood level of bile acids, a disease or disorder associated with a pathological blood level of hydrophobic bile acids or a pre-existing disease or disorder as described herein; or (iv) a combination of at least two of (I) to (ill).

[0152] Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0153] BRIEF DESCRIPTION OF THE DRAWINGS

[0154] In the appended drawings:

[0155] FIGs. 1A-B: (FIG. 1A) Rapid equilibrium dialysis (RED) plate in the style of a 96-well plate including plate inserts consisting of the sample chamber (red) and buffer chamber (white) separated by a membrane with a cut-off weight of 12 kDa. (FIG. 1 B) The RED system is filled with the equal concentration of bile acid in both chambers, whereas VS- 01 and human albumin are only added to the buffer chamber. During incubation VS-01 and human albumin will bind bile acids, which leads to a flow of bile acid from the sample chamber into the buffer chamber to equilibrate the bile acid concentration in the system. After incubation a sample can be taken out of the sample chamber to investigate the bile acid binding.

[0156] FIGs. 2A-B: The cholic acid binding as a synergistic effect of human albumin (9 mg / mL) and VS-01 (5 mM) (FIG. 2A) or VS-01 (8 mM) (FIG. 2B) is shown in this bar plot by showing the cholic acid binding % of human albumin (left) and VS-01 (middle) alone and in combination (right) (n=2 / condition).

[0157] FIG. 3: The glycocholic acid binding as a synergistic effect of human albumin (9 mg / mL) and VS-01 (5 mM) is shown in this bar plot by showing the glycocholic acid binding % of human albumin (left) and VS-01 (middle) alone and in combination (right) (n=2 / condition). FIG. 4: The glycochenodeoxycholic acid binding as a synergistic effect of human albumin (9 mg / mL) and VS-01 (2 mM) is shown in this bar plot by showing the glycochenodeoxycholic acid binding % of human albumin (left) and VS- 01 (middle) alone and in combination (right) (n=2 / condition).

[0158] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0159] The present disclosure is illustrated in further detail by the following non-limiting examples.

[0160] EXAMPLE 1 : Material and Methods

[0161] VS-01 Liposome formulation

[0162] Liposome aqueous suspension (LAS)

[0163] A lipid blend composed of 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Lipoid), cholesterol (Sigma-Aldrich) and N-(carbonyl-methoxy-polyethylene glycol 2000)-1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine (sodium salt of mPEG-DSPE, Lipoid) in a molar ratio of 85.5: 14:0.5 (100 mg / g lipids) equivalent to a weight % ratio of 90.2:7.78:2.01 , respectively was prepared by the spray drying method. 614 mg of DPPC, 53.0 mg of cholesterol and 14.0 mg of DSPE-PEG were co-dissolved in ethanol > 96% (v / v) prior spray drying and heated up to 45°C under stirring until complete dissolution of the lipids. After a filtration step (using a 0.45 m filter), the warm lipid solution was spray-dried with a high-pressure nozzle using liquid nitrogen as drying gas and a feed rate between 10 and 20 kg / hr and an outlet temperature of 55 - 65°C. Ethanol evaporated during the spray-drying process. The dried lipid blend was hydrated with ultra-pure water (aqueous medium) (lipids concentration = 100 mg / mL144 pM / mL) while heating up to 60°C to ensure that the product was above the lipid hydration temperature (55°C) and stirred 2 hours. The hydrated blend was then degassed using a vacuum pump and finally sterilized in sealed bottles by autoclaving 20 min at 121 °C. Bottles were stored at 2-8°C. Liposomes having an average diameter between about 10 pm and 15 pm were obtained.

[0164] Citric acid anhydrous solution (CAS)

[0165] A citrate buffer 600 mM (pH 2.1 , 1041 mOsm / l) containing citric acid (600 mM; 115 g / L), sodium chloride (143 mM ; 8.36 g / L), sodium hydroxide (97.5 mM; 3.90 g / L), and magnesium chloride hexahydrate (12 mM; 2.44 g / L) in water (acidic and hyperosmotic buffer) was prepared as follows: Each solid ingredient was accurately weighed. The appropriate amount of water for injection was added and the mixture was stirred at room temperature until complete dissolution of all the salts. The acidic solution was then 0.2 pm filtered before it was filled into 50 mL-COC injection bottles. Bottles of citric acid solution (CAS) were terminally steam sterilized in their final container utilizing the same reference conditions as the Ph. Eur. Chapter 5.1 .1 (>121 °C, >15 min in all units).

[0166] Sterile xylitol alkaline solution (XAS)

[0167] A neutralization solution (21.3 mg / mL, pH = 12.6, 450 mOsm / l) (neutralizing aqueous solution) in a suitable format (e.g., infusion bag) comprising an osmotic agent, xylitol, in an alkaline salt solution was prepared as follows. Xylitol (152 g / mol; 140 mM; 21.3 g / L), sodium chloride (58.4 g / mol; 92.0 mM; 5.38 g / L), sodium hydroxide (40.0 g / mol; 50.0 mM; 2.00 g / L), potassium chloride (74.6 g / mol; 2.00 mM; 0.149 g / L), and calcium chloride (147 g / mol; 0.500 mM; 0.0735 g / L) were added to water for injection. Each solid ingredient was accurately weighed. The appropriate amount of water for injection was added and the mixture stirred at room temperature until complete dissolution of all the salts. The alkaline solution was then 0.2 pm filtered before it was filled into an infusion bag. The bag was terminally steam sterilized in its final container utilizing the same reference conditions as the Ph. Eur. Chapter 5.1.1 (>121 °C, >15 min in all units).

[0168] Liposome reconstitution

[0169] 2.104 g of the above-described sterile citric acid anhydrous solution (CAS) were transferred to 4.215 g of the abovedescribed liposomal aqueous suspension (LAS) in a glass vial. The vial was then placed on a roller mixer (Benchmark Scientific, Model: TubeRoller) (60 rpm) for 10 min at RT. A liposome composition was produced containing in a solution of the citric acid anhydrous solution, the liposomes enclosing the citric acid anhydrous solution.

[0170] The transmembrane pH-gradient was generated by neutralizing the external acid medium of the liposome composition by transferring 4.861 g of the liposome composition into a 50 mL Falcon tube with 45.701 g of the abovedescribed sterile xylitol alkaline solution (XAS). The mixture of liposome composition and xylitol alkaline solution was inverted 5 times and incubated for 15 min on a roller mixer (60 rpm). Immediately after, pH was measured and was within specification (pH= 5-8).

[0171] The resulting multilamellar liposomes suspension (including the transmembrane pH-gradient liposomes) containing 18.4 mM citric acid anhydrous, 9.3 mM total lipid, at pH 6.5-6.6 and about 310 mOsm / l (VS-01) (liposomal suspension) was used for the assays presented herein and in preclinical studies.

[0172] Final transmembrane pH gradient liposomes concentrations of 2, 5 and 8 mM were tested with the RED system.

[0173] Human Albumin

[0174] A stock solution of human albumin (Recombinant, lyophilized powder, low endotoxin, suitable for cell culture, Sigma- Aldrich, SAE0160-5G, Source: SLCQ8521) in HEPES buffer (pH 7.40, 20 mM, 350 mOsm / kg) with a concentration of 180 mg / mL was prepared. Final human albumin concentrations of 6 and 9 mg / mL were tested with the RED system.

[0175] Cholic Acid

[0176] A stock solution of cholic acid (408.6 Da) (Sodium cholate hydrate, Sigma-Aldrich, Art. Nr.: C9282-25G, Source: SLCR3084, PCode: 1003629888) was prepared in HEPES buffer (pH 7.40, 20 mM, 350 mOsm / kg) with a concentration of 1518.3 piM. A final concentration of 50 piM was used in the RED system for the experiments.

[0177] Glycocholic Acid A stock solution of glycocholic acid (465.6 Da) (Sodium glycocholate hydrate, Sigma-Aldrich, Art. Nr.: G7132-100MG, Source: SLCH8812, PCode: 1003611 123) in HEPES buffer (pH 7.40, 20 mM, 350 mOsm / kg) with a concentration of 1220.5 piM was prepared. Final concentration of 50 piM was used in the RED system for the experiments.

[0178] Glycochenodeoxycholic Acid

[0179] A stock solution of glycochenodeoxycholic acid (449.6 Da) (Sodium Glycochenodeoxycholate, Merck, Art. Nr.: G0759-100MG, Source: SLCK1470, PCode: 1003349072) was prepared in HEPES buffer (pH 7.40, 20 mM, 350 mOsm / kg) with a concentration of 1392.6 piM. A final concentration of 50 piM was used in the RED system for the experiments.

[0180] Binding assays

[0181] The binding of cholic acid (CA), glycocholic acid (GCA) and glycochenodeoxycholic acid (GCDCA) by human albumin (6 and 9 mg / mL) and the transmembrane pH gradient liposomes (liposomal suspension) described above in Example 1 (2, 5 & 8 mM) individually or in combination was investigated using an in vitro diffusion system, namely the Rapid Equilibrium Dialysis System (RED) from Thermo Fisher Scientific (Rapid Equilibrium Dialysis System (RED), Device Single-Use with Inserts, 12 kDa, Thermo Fisher Scientific, Cat. Nr.: 901 12, Lot: ZB421 1611 & YK41 14551 & YJ4067311 , FIGs. 1A-B).

[0182] The RED device is a diffusion system which consists of two chambers, a sample chamber and a buffer chamber, which are separated by a membrane with a cut-off weight of 12 kDa (FIG. 1A). This cut-off weight allows bile acids used (i.e. 0.409-0.466 kDa) to move freely through the membrane leading to an equilibration of the bile acid concentration in both chambers (FIG. 1 B), whereas human albumin (66.5 kDa) and VS-01 (15 pirn) are retained. The bile acid concentration in both chambers was equal (50 piM), whereas human albumin and VS-01 were only loaded in the buffer chamber.

[0183] The RED system was loaded by adding, in sequence, HBS (HEPES buffer saline (pH 7.40, 20 mM, 350 mOsm / kg), the corresponding bile acids described above, the transmembrane pH gradient liposomes (liposomal suspension) as described above in Example 1 , and finally human albumin as described above in Example 1. During incubation, VS- 01 and human albumin bound bile acids in the buffer chamber. All measurements were performed in duplicate. The RED system plate was closed with sealing tape to prevent water evaporation during incubation. The plate was incubated at 37 °C for 4h in an incubator (HERAtherm™ Oven, Thermo Scientific), where it was placed on an orbital shaker (Battery Shaker KM2, Edmund Buhler GmbH) at shaking rate of 200 1 / min. After 4h samples of 200 piL were taken out of the sample chamber and used for bile acid quantification. All combinations were incubated at 37 °C for 4h.

[0184] Bile acid quantification

[0185] For bile acid quantification 200 piL were taken out from the sample chamber after incubation at 37 °C for 4h. Two independent reference samples of bile acid concentration at start (tO) were prepared following the sample chamber loading instructions. Bile acids were quantified using an enzymatic kit (RX series TBA, Randox™, ref: BI3863, lot: 663507) and a Randox Daytona+™ instrument from Randox. The binding was calculated by subtracting the final bile acid concentration (t4h) in the sample chamber from the bile acid concentration in the sample chamber at start (tO).

[0186] Synergistic Effect Calculation: Bliss Independence Model

[0187] The Bliss independence model is one of the most popular models, assuming both drugs contribute independently to the observed effect. The drug effects are expressed in the form of a probability using the calculation index (Cl), indicating a synergistic effect if Cl is below 1 , an additive effect if Cl equals 1 and an antagonism effect if Cl is above 1. The calculation index Cl is calculated according to equation below, where EA and EB are the observed effects of drug A and B alone, and EAB is the observed effect of drug A and B in combination:

[0188] EXAMPLE 2: A combination of liposomes and albumin synergistically bind to cholic acid

[0189] Measurements assessing binding of cholic acid (CA) to VS-01 (2, 5, 8 mM) and human albumin (6 and 9 mg / mL) alone and in combination with VS-01 (2, 5, 8 mM) were performed with a cholic acid concentration of 50 piM at start. The cholic acid binding rate is shown in Table II below. It was observed that VS-01 alone binds cholic acid at high concentration (8 mM, 6.6% binding rate), while albumin alone was found to bind cholic acid in a dose-dependent manner, with best binding rates obtained at 9 mg / mL albumin (11 .8%). It was shown that the combination of albumin and VS-01 further improved cholic acid binding. For example, the binding rates obtained at medium concentration of VS-01 (5 mM) and human albumin (9 mg / mL) increased from 0.4% and 11.8%, respectively, up to 19.3% in combination, showing a synergistic effect of VS-01 and human albumin in combination (FIG. 2A). Also, the binding rates obtained at high concentrations of VS-01 (8 mM) and human albumin (9 mg / mL) increased from 6.6% and 11.8%, respectively, up to 31.7% in combination, showing a synergistic effect of VS-01 and human albumin in combination (FIG. 2B). Table III shows that 6 & 9 mg / mL human albumin concentrations with VS-01 have a synergistic effect on the cholic acid binding according to the Bliss independence model. Table II Cholic acid binding % obtained with VS-01 and human albumin alone or in combination

[0190] Table III Synergistic effect of all tested combinations of VS-01 and albumin on cholic acid calculated using the Bliss Independence model (Bliss C1 value)

[0191] EXAMPLE 3: A combination of liposomes and albumin synergistically bind to glycocholic acid

[0192] Measurements assessing binding of glycocholic acid (GCA) to VS-01 (2, 5, 8 mM) and human albumin (6 and 9 mg / mL) alone and in combination were performed with a glycocholic acid concentration of 50 piM at start. The glycochenodeoxycholic acid binding rate is shown in Table IV below. It was observed that VS-01 alone binds glycocholic acid in a dose-dependent manner, with best binding rate obtained at 8 mM (11.8%), while albumin alone was found to bind glycocholic acid to similar levels at 6-9 mg / mL (4.9%, and 4.3%, respectively). It was shown that the combination of human albumin and VS-01 further improved glycocholic acid binding. For example, the binding rates obtained at 5 mM VS-01 and 9 mg / mL human albumin concentration increased from 3.2% and 4.3%, respectively, up to 8.6% in combination, showing a synergistic effect of VS-01 and human albumin in combination (FIG. 3). Table V shows this synergistic effect calculated according to the Bliss independence model.

[0193] Table IV Glycocholic acid binding % obtained with VS-01 and human albumin alone or in combination

[0194] Table V Synergistic effect of all tested combinations of VS-01 and albumin on glycocholic acid calculated using the Bliss Independence model (Bliss C1 value)

[0195] EXAMPLE 4: A combination of liposomes and albumin synergistically bind to glycochenodeoxycholic acid

[0196] Measurements assessing binding of glycochenodeoxycholic acid (GCDCA) to VS-01 (2, 5, 8 mM) and human albumin (6 and 9 mg / mL) alone and in combination were performed with a glycochenodeoxycholic acid concentration of 50 piM at the start. The glycochenodeoxycholic acid binding rate is shown in Table VI below. It was observed that VS-01 alone binds glycochenodeoxycholic acid in a dose-dependent manner, with best binding rate obtained at 8 mM (31 %), while albumin alone was found to bind glycochenodeoxycholic acid at higher levels at 6 mg / mL (28.2%). It was shown that the combination of human albumin and VS-01 further improved glycochenodeoxycholic acid binding. For example, the binding rates obtained at 2 mM concentration of VS-01 and 9 mg / mL human albumin concentration increased from 10.1 % and 14.4% respectively, up to 35.5% in combination, showing a synergistic effect of VS-01 and human albumin in combination (FIG. 4). Table VII shows that the combination of VS-01 2 mM or 5 mM and human albumin 9 mg / mL has a synergistic effect on the glycochenodeoxycholic acid binding according to the Bliss independence model.

[0197] Table VI. Glycochenodeoxycholic acid binding % obtained with VS-01 and human albumin alone or in combination

[0198] Table VII Synergistic effect of all tested combinations of VS-01 and albumin on glycochenodeoxycholic acid calculated using the Bliss Independence model (Bliss C1 value)

[0199] A combination of VS-01 (2 mM) and 6 mg / mL or 9 mg / mL albumin was also synergistic in sequestering CDCA (data not shown).

[0200] To summarize, liposomes of the present disclosure and albumin alone can bind hydrophobic bile acids (binding rates for VS-01 : GCDCA>, GCA> CA, binding rates for albumin: GCGCA>CA>GCA). The combination of liposomes and albumin further enhances the percentage of captured bile acid with best synergy obtained at 9 mg / mL human albumin in combination with any concentration of VS-01 for cholic acid (CA), with 5 mM VS-01 for glycocholic acid (GCA) and with 2 mM or 5 mM VS-01 for glycochenodeoxycholic acid (GCDCA).

[0201] EXAMPLE 5: Sequential in vivo intraperitoneal administration of liposomes and albumin in animal model of liver impairment

[0202] First, a therapeutic dose transmembrane pH-gradient liposomes VS-01 (18.4 mM citric acid and 9.3 mM total lipid) is injected into the peritoneal cavity of the rats (e.g., healthy adult rats), followed by a dwell time of four hours. The dialysate including VS-01 is thereafter removed. The cavity is then rinsed with either (a) an albumin-containing wash solution (e.g., Flexbumin™) containing one of various albumin concentrations (VS-01 + albumin groups); or (b) a saline (VS-01 + saline group) for a dwell time of four hours and removed thereafter. Peritoneal fluid and blood samples are withdrawn at pre-dose, at various preallotted times during VS-01 dwell time, during albumin or saline wash, and at post-dose. These samples are quantified for albumin, and bile acids.

[0203] Second, the optimal dose and dwell time of albumin so identified are used in a washing test after administration of VS-01 in ACLF animals (CCI4 rat model (rats sustaining acute liver injury via carbon tetrachloride (CCI4)) and / or LPS rat model). A therapeutic dose of VS-01 is thus injected into the peritoneal cavity of the animal models followed by a 4-hour dwell time. VS-01 is subsequently removed, and the cavity rinsed with a dose of the albumin-containing wash solution. The study comprises two groups VS-01 + saline vs. VS-01 + albumin. Peritoneal fluid and blood samples are withdrawn at pre-dose, during VS-01 dwell time, during albumin wash, and at post-dose. These samples are quantified for metabolites, such as but not limited to total bile acids, total bilirubin and ACLF-related metabolites including cholic acid, glycocholic acid and glycochenodeoxycholic acid.

[0204] EXAMPLE 6: Intraperitoneal administration of combination of liposomal suspension and albumin solution in animal model of liver impairment

[0205] A transmembrane pH gradient liposomes solution (VS-01) (liposomal suspension) alone, an albumin solution alone or a combination of VS-01 and albumin in solution are administered intraperitoneally to a rat model of ACLF. ACLF- related metabolites / toxins are then dosed in the blood and peritoneal fluid of each group of animals. Improvements in ACLF-induced levels of these toxins are compared between groups.

[0206] EXAMPLE 7: Combination of liposomal suspension and albumin for the treatment of ascites

[0207] Patients undergoing large volume paracentesis (ascites removal) represent a subgroup of subjects that could benefit from a treatment with transmembrane pH gradient liposomes (VS-01). Albumin intravenous infusion or intraperitoneal administration (8g / L of ascites removed) is performed before or simultaneously with VS-01 intraperitoneal administration (i.p).

[0208] The scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

CLAIMS:

1. Combination of a liposomal suspension, and of albumin, for use as bile acid sequestrants, wherein the use comprises (a) intraperitoneally administering the liposomal suspension to a subject in need thereof; and (b) intravenously or intraperitoneally administering the albumin to the subject; and (c) removing a dialysate containing liposomes and at least one bile acid from the subject, wherein the at least one bile acid comprises cholic acid, glycocholic acid, glycochenodeoxycholic acid or a combination of at least two thereof.

2. The combination for use of claim 1 , wherein in (a) the liposomal suspension is in a dose of between about 2 mM to about 10mM or of between about 2 mM to about 9.5 mM.

3. The combination for use of claim 1 or 2, wherein in (b) the albumin is in a dose of between about 6 mg / mL to about 10 mg / mL, preferably between about 8 mg / mL to about 10 mg / mL, most preferably about 9 mg / mL.

4. The combination for use of any one of claims 1 to 3, wherein (a) and (b) are simultaneous.

5. The combination for use of any one of claims 1 to 4, wherein the administering in (b) is intraperitoneal.

6. The combination for use of any one of claims 1 to 5, wherein the liposomes contain a hydroxy acid, preferably citric acid, most preferably citric acid anhydrous.

7. The combination for use of claim 6, wherein the liposomes contain about 200 nM citric acid anhydrous.

8. The combination for use of any one of claims 1 to 7, wherein the liposomes’ lipid bilayer comprises at least one phospholipid as main constituent.

9. The combination for use of claim 8, wherein the at least one phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), preferably in a range of 60 mol% to 90 mol%.

10. The combination for use of any one of claims 1 to 9, wherein the liposomes’ lipid bilayer comprises cholesterol, preferably in a range of 10 to 40 mol%.

11. The combination for use of claim 10, wherein the liposomes’ lipid bilayer further comprises 1 ,2-distearoyl-sn- glycero-3-phosphoethanol-amine-N-[methoxy(PEG)-2000] (DSPE-PEG), preferably in a range of 0.2 to 5 mol%.

12. The combination for use of any one of claims 1 to 11 , wherein the bilayer of the liposomes contains dipalmitoylphosphatidylcholine (DPPC), cholesterol and 1 ,2-distearoyl-sn-glycero-3-phosphoethanol-amine- N-[methoxy(PEG)-2000] (DSPE-PEG) at 85.5: 14:0.5 mol%, and the liposomes’ inner compartment contains citric acid anhydrous.

13. The combination for use of any one of claims 1 to 12, wherein the liposomes have an average diameter between about 8 pm and 15 pm.

14. The combination for use of any one of claims 1 to 13, wherein the liposomal suspension contains (I) xylitol, (II) sodium chloride, (ill) sodium hydroxide, (iv) potassium chloride, (v) calcium chloride or (vii) any combination of at least two of (I) to (v), preferably the combination comprises all of (I) to (v).

15. The combination for use of any one of claims 1 to 14, wherein the subject is a human, preferably a subject having ACLF.

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